title: A comparison of single molecule and amplification based sequencing of cancer transcriptomes.
abstract: The second wave of next generation sequencing technologies, referred to as single-molecule sequencing (SMS), carries the promise of profiling samples directly without employing polymerase chain reaction steps used by amplification-based sequencing (AS) methods. To examine the merits of both technologies, we examine mRNA sequencing results from single-molecule and amplification-based sequencing in a set of human cancer cell lines and tissues. We observe a characteristic coverage bias towards high abundance transcripts in amplification-based sequencing. A larger fraction of AS reads cover highly expressed genes, such as those associated with translational processes and housekeeping genes, resulting in relatively lower coverage of genes at low and mid-level abundance. In contrast, the coverage of high abundance transcripts plateaus off using SMS. Consequently, SMS is able to sequence lower- abundance transcripts more thoroughly, including some that are undetected by AS methods; however, these include many more mapping artifacts. A better understanding of the technical and analytical factors introducing platform specific biases in high throughput transcriptome sequencing applications will be critical in cross platform meta-analytic studies.
authors.affiliation: Michigan Center for Translational Pathology, University of Michigan, Ann Arbor, Michigan, United States of America.
authors.forename: Lee T
authors.initials: LT
authors.lastname: Sam
authors.forename: Doron
authors.initials: D
authors.lastname: Lipson
authors.forename: Tal
authors.initials: T
authors.lastname: Raz
authors.forename: Xuhong
authors.initials: X
authors.lastname: Cao
authors.forename: John
authors.initials: J
authors.lastname: Thompson
authors.forename: Patrice M
authors.initials: PM
authors.lastname: Milos
authors.forename: Dan
authors.initials: D
authors.lastname: Robinson
authors.forename: Arul M
authors.initials: AM
authors.lastname: Chinnaiyan
authors.forename: Chandan
authors.initials: C
authors.lastname: Kumar-Sinha
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Maher
mesh_terms: D001483:Base Sequence; D015982:Bias; D020869:Gene Expression Profiling / Q000379:methods*; D015972:Gene Expression Regulation, Neoplastic; D050939:Gene Fusion; D052138:Genes, Neoplasm / Q000235:genetics; D006801:Humans; D009369:Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D012333:RNA, Messenger